Phosphoinositides present in the erythrocyte membrane have a unique function when compared to many other cell types. In the erythrocyte the phosphoinositides play a structural role in contrast to the more commonly observed signal transduction function. Specifically, these lipids have been implicated in the control of the native biconcave shape of the erythrocyte as well as serving as a component in the skeletal complex between band 4.1 and glycophorin. With this background, the focus of the present proposal is to understand the physiological function and enzymology of the phosphoinositides in erythrocytes. The first goal is to purify and characterize phosphatidylinositol 4-phosphate kinase. This enzyme, which has been shown to be a component of the erythrocyte skeleton, is a critical element in controlling the level of phosphatidylinositol 4,5-bisphosphate in the membrane. Therefore, the enzyme will be purified, its kinetic properties examined both on and off the skeleton, and the binding site for this enzyme on the skeleton will be identified. The second goal of this proposal is to examine the phosphoinositide metabolism of senescent erythrocytes. Mature erythrocytes cannot synthesize additional phosphatidylinositol so that the possibility that they have a decreasing level of this lipid during their life span is of particular interest with regards to the viability of aged erythrocytes. A recently developed procedure for the isolation of senescent erythrocytes will be utilized; this procedure involves the biotinylation of rabbit erythrocytes. The reinfusion of the derivatized cells and their eventual recovery, after in vivo aging, by attachment to an avidin support. The third goal of the project is to examine the maintenance of distinct pools of phosphoinositides within the erythrocyte membrane. Preliminary work has shown that a sizable portion of the erythrocyte phosphoinositides are associated with the skeleton and that the exchange between this pool and the bulk of the phosphoinositides is slow enough to be readily studied. The final goal of the project is to examine patients with undefined hemolytic anemia for the presence of abnormalities in the phosphoinositide pathways.